The healing of skin defects progresses through three general phases: (i) inflammation, (ii) wound cell migration and mitosis, and (iii) extracellular matrix production and remodeling. The ordered sequence of these events is thought to be orchestrated by interactions among cells, growth factors, and extracellular matrix proteins. A crucial step of skin wound healing is epidermal regeneration (i.e., re-epithelialization). Besides interfollicular epidermal keratinocytes from the wound edges, the outer root sheath (ORS) cells from residual hair follicles also contribute to this process (see e.g., Eisen et al., 15 J. Invest. Dermatol. 145–155 (1955)). The ORS of hair follicles is comprised largely of undifferentiated keratinocytes that encompass the cylindrical structures of the hardened inner root sheath and the hair shaft (see e.g., Montagna & Parakkal, In: The Structure and Function of Skin 172–258 (Academic Press New York, N.Y., 1974)). Recent literature has also indicated that ORS cells are at a lower level of commitment to differentiation than the basal interfollicular keratinocytes (see e.g., Coulombe et al., 109 J. Cell Biol. 2295–2312 (1989); Limat et al., 194 Exp. Cell Res. 218–227 (1991); Limat et al., 275 Cell Tissue Res. 169–176 (1994)), and label-retaining cells have been detected in the animal as well as the human ORS region near the bulge area which possibly represent stem cells for skin epithelial tissues (see e.g., Cotsarelis et al., 61 Cell 1329–1337 (1990); Kobayashi et al., 90 Proc. Nat. Acad. Sci. USA 7391–7395 (1993); Yang et al., 105 J. Invest. Dermatol. 14–21 (1993); Rochat et al., 76 Cell 1073–1076 (1994); Moll, 105 J. Invest. Dermatol. 14–21 (1995)). Additionally, human ORS cells which are isolated from plucked anagen scalp hair follicles can be expanded extensively in vitro (see e.g., Weterings et al., 104 Brit. J. Dermatol. 1–5 (1981); Limat & Noser, 87 J. Invest. Dermatol. 485–488 (1986); Imcke et al., 17 J. Am. Acad. Dermatol. 779–786 (1987): Limat et al., 92 J. Invest. Dermatol. 758–762 (1989)). Under conventional submerged culture conditions, ORS cells resemble interfollicular epidermal keratinocytes by both morphologic and biochemical (e.g., keratin profiles) criteria (see e.g., Stark et al., 35 Differentiation 236–248 (1987); Limat et al., 92 J. Invest. Dermatol. 758–762 (1989); Limat et al., 642 Ann. N.Y. Acad. Sci. 125–147 (1991)). In organotypic co-cultures with human dermal fibroblasts (i.e., under conditions mimicking the epidermal environment), ORS cells with respect to histological, immunohistological, ultrastructural and biochemical criteria develop a stratified epithelium reminiscent of regenerating epidermis (see e.g., Lenoir et al., 130 Dev. Biol. 610–620 (1988); Limat et al. 194 Exp. Cell Res. 218–227 (1991); Limat et al., 642 Ann. N.Y. Acad. Sci. 125–147 (1991)). If such organotypic cultures are grafted onto nude mice, ORS cells form a regular neo-epidermis that is under homeostatic control (see e.g., Limat et al, 59 Transplantation 1032–1038 (1995)). Thus, human ORS cells are of considerable interest for clinical application.
In the previous decade, interest has focused on the use of cultured epithelial cells for wound coverage. First, sheets of cultured autologous interfollicular keratinocytes were grafted successfully on acute wounds, mainly in the treatment of larger third degree burns (see e.g., O'Connor et al., 1 Lancet 75–78 (1981); Compton et al., 60 Lab. Invest. 600–612 (1989)) but also of epidermolysis bullosa (see e.g., Carter et al. 17 J. Am. Acad. Dermatol. 246–250 (1987)), pyoderma gangrenosum (see e.g., Dean et al. 26 Ann. Plast. Surg. 194–195 (1991); Limova & Mauro, 20 J. Dermatol. Surg. Oncol. 833–836 (1994)), and wounds after excision of giant congenital nevi (see e.g., Gallico et al., 84 J. Plast. Reconstr. Surg. 1–9 (1989)) or separation of conjoined twins (see e.g., Higgins et al., 87 J. Royal Soc. Med. 108–109 (1994)).
In contrast to the treatment of such acute wounds, the grafting of chronic wounds (e.g., leg ulcers) with cultured keratinocytes has been much less successful. Allografts do not result in a permanent “take” (see e.g., Fabre. 29 Immunol. Lett. 161–166 (1991)) and thus may be classified as a “quite effective but expensive biological dressing” (see Phillips et al., 21 J. Am. Acad. Dermatol. 191–199 (1989). A reproducible, major definite “take” of autologous keratinocyte grafted by various modalities including: sheets of submerged keratinocyte cultures consisting of only a few, noncornified cell layers (Hetton et al., 14 J. Am. Acad. Dermatol. 399–405 (1986); Leigh & Purkis, 11 Clin. Exp. Dermatol. 650–652 (1986); Leigh et al, 117 Brit. J. Dermatol. 591–597 (1987); Harris et al., 18 Clin. Exp. Dermatol. 417–420 (1993)), trypsinized single cells attached to collagen-coated dressings (Brysk et al., 25 J. Am. Acad. Dermatol. 238–244 (1991)), skin equivalents (Mol et al., 24 J. Am. Acad. Dermatol. 77–82 (1991)) has yet to be convincingly documented within the scientific literature. The same lack of quantitative findings also holds true for various reports on the grafting of freshly isolated, autologous interfollicular keratinocytes (Hunyadi et al., 14 J. Dermatol. Surg. Oncol. 75–78 (1988)) or ORS cells (Moll et al., 46 Hautarzt 548–552 (1995)) fixed to the wound bed by the use of a fibrin glue. However, it should be noted that the disadvantages of the bovine serum used during cultivation of the keratinocytes may contribute to reduced “take” rate, due to the fact that it resists in keratinocytes (see e.g., Johnson et al., 11 J. Burn Care Rehab. 504–509 (1990)).
DE-A-19651992 describes the culture of outer root sheath cells in 10–15% autologous or homologous serum to produce dermal equivalents. The dermal equivalents may be seeded on hyaluronic acid membranes or other biodegradable material prior to transplantation in order to optimise handling.
Lenoir-Viale, M. C. (Arch. Dermatol. Res. 1993, 285: pages 197–204) describes the in vitro preparation of a reconstructed epidermis from the outer root sheath of human hair follicles. The reconstructed epidermis is described as a valuable and promising tool for pharmacological studies and may represent a model of wound-healing.
Limat, A. (J. of Investigative Dermatology 2000, Nov. 7, pages 128–134) describes the culturing of hair follicles (hair bulbs and infundibular parts removed) to generate epidermal equivalents and the use thereof for treating chronic leg ulcers.